1. Field of Invention
This invention relates to DNA vaccines, specifically to improved DNA vaccines that induce strong antigen-specific humoral and cellular immune responses.
2. Description of Prior Art
DNA immunization, the inoculation of plasmid DNA encoding a microbial or tumor antigen, is a recent addition to vaccine technology (Donnelly J. J. et al, Ann. Rev. Immunol. 15: 617–648, 1997; Letvin N. L., Science 280: 1875–1879, 1998). Both cellular and humoral immune responses occur after DNA vaccination, and protective immunity against microbial challenge is sometimes induced in experimental animals (Ulmer J. B. et al, Vaccine 12: 1541–1544, 1994; Yokoyama M. et al, J. Virol. 69: 2684–2688, 1995; Xiang Z. Q. et al, Virology 199: 132–140, 1994; Sedegah M. et al, Proc. Natl. Acad. Sci. USA 91: 9866–9870, 1994; Montgomery D. L. et al, DNA Cell Biol. 12: 777–783, 1993). T cell responses, including CD8+ cytotoxic T lymphocyte (CTL) and CD4+ T helper cells, can be stimulated by DNA vaccination in response to antigenic peptides presented by class I and class II MHC molecules (Whitton J. L. et al, Vaccine 17: 1612–1619, 1999). Endogenous protein synthesis allows presentation of foreign antigenic peptides by MHC class I, whereas uptake of soluble protein by APC is required for presentation of peptides by MHC class II. Both arms of the immune response can therefore be induced after DNA vaccination, but the pathways for antigen processing and presentation are distinct for peptides presented by MHC class I or MHC class II. This conclusion is derived from experiments using DNA encoding ubiquitinated protein that is rapidly targeted to intracellular degradation by proteosomes. Ubiquitinated antigen that was degraded so rapidly that intact protein could not leave the cell led to enhanced production of CTL in vivo, but completely eliminated antibody production (Rodriguez F. et al, J. Virol. 71: 8497–8503, 1997; Wu Y. and Kipps T. J., J. Immunol. 159: 6037–6043, 1997). Thus a major limitation of DNA vaccines is their inability to induce strong and sustained humoral immune responses. Strategies for optimization of the cellular immune response to DNA vaccines that do not reduce humoral immune responses are needed.
DNA vaccines for HIV-1 have been tested in animal models and found to induce an immune response that provides protection against challenge only when the virulence of the viral isolate is low. In benign challenge models, chimpanzees were protected from live virus exposure by vaccination with plasmid DNA or by subunit antigens or peptides (Boyer J. D. et al, Nat. Med. 3:526–532, 1997; Kennedy R. C., Nat. Med. 3: 501–502, 1997). However, when highly virulent SIV was tested in rhesus macaques, DNA vaccination was not protective and could only achieve a reduction in virus load even when multiple doses of DNA were inoculated through multiple routes (Lu S. et al, J. Virol. 70: 3978–3991, 1996). Therefore, enhancing the immune response to DNA immunization is an important goal of current AIDS vaccine research. Enhancing the immune response to other DNA vaccines is also desirable in order to provide protection when infected with highly virulent organisms or with a high infectious dose, and to provide long lasting protection. Enhancing the immune response to DNA vaccines encoding tumor antigens is also important for maximizing the anti-tumor response.
One strategy that has been tested is to prime with a DNA vaccine followed by boosting with protein antigen. However, this approach requires construction of multiple vaccines for the same infection or disease, and depends upon multiple injections given in a precise order. It would be desirable to induce protective immunity without needing multiple forms of a vaccine, and without requiring alternating injections of DNA and protein.
Chemical and genetic approaches to enhance the immune response to DNA vaccines have been studied. Chemical adjuvants with some activity include monophosphoryl lipid A (Sasaki S. et al, Infect. Immun. 65: 3520–3528, 1997), saponin QS-21 (Sasaki S et al, J. Virol. 72: 4931–4939, 1998), mannan-coated liposomes (Toda S et al, Immunology 92: 111–117, 1997), and the aminopeptidase inhibitor ubenimex (Sasaki S et al, Clin. Exp. Immunol. 11: 30–36, 1998). Each of these adjuvants modestly enhanced both antibody titers and CTL activity after DNA vaccination in mice. Although the mechanism of action of chemical adjuvants is not fully elucidated, they seem to work by induction of cytokines that amplify responses, by recruitment of macrophages and other lymphoid cells at sites of DNA administration, or by facilitating entry of DNA into host cells (Sasaki S. et al, Anticancer Research 18: 3907–3916, 1998). Several genetic approaches to enhancing responses to DNA vaccines have been tested, including administration of a gene encoding a cytokine (IL2, IL 12, GM-CSF, TCA3, MIP-1α) (Chow Y.-H. et al, J. Virol. 71: 169–178, 1997; Hwee Lee A. et al, Vaccine 17: 473–479, 1998; Tsuji T. et al, Immunol. 158: 4008–4014, 1997; Rodriguez D. et al, Gen. Virol. 80: 217–223, 1999; Tsuji T. et al, Immunology 90: 1–6, 1997; Lu Y. et al, Clin. Exp. Immunol. 115: 335–341, 1999) or a costimulatory adhesion receptor (CD86, CD58, CD54) (Tsuji T. et al, Eur. J. Immunol. 27: 782–787, 1997; Kim J. J. et al, J. Clin. Invest. 103: 869–877, 1999; Iwasaki A. et al, J. Immunol. 158: 4591–4601, 1997). Each of these cytokine and adhesion receptor genes increased immune responses to DNA vaccination, with some treatments enhancing CTL generation only, and some enhancing both CTL and antibody production. However, the levels of enhancement of the immune response to DNA vaccination obtained from these approaches are modest and not sustained, so it is important to find additional ways to enhance the immune response to DNA vaccines.
The CD40 receptor must be activated for an effective cellular or humoral immune response after exposure to antigen (Grewal I. S., and Flavell R. A., Annu. Rev. Immunol 16: 111–135, 1998). This conclusion is derived from multiple findings, including the phenotype of patients with hyper IgM (HIGM) syndrome that results from CD154 genetic defects (Aruffo A. et al, Cell 72: 291–300, 1993; Fuleihan R. et al, Proc. Natl. Acad. Sci. USA 90: 2170–2173, 1993; Korthauer U. et al, Nature 361: 539–541, 1993), the phenotype of mice with CD40 or CD154 gene disruption (Grewal I. S. et al, Science 273: 1864–1867, 1996; Kawabe T. et al, Immunity 1: 167–178, 1994; Renshaw B. et al, J. Exp. Med. 180: 1889–1900, 1994; Xu J. et al, Immunity 1: 423–431, 1994), and the effects of actively blocking CD40 in vivo using inhibitory antibodies to CD154 (Durie F. H. et al, Science 261: 1328–1330, 1993; Foy T. M. et al, J. Exp. Med. 178: 1567–1575, 1993; Foy T. M. et al, J. Exp. Med. 180: 157–163, 1994; Durie F. H. et al, J. Clin. Invest. 94: 1333–1338, 1994; Gerritsse K. et al, Proc. Nat. Acad. Sci. USA 93: 2499–2504, 1996). CD40 is expressed in several cell lineages, including B cells, dendritic cells, monocytes, epithelial cells, and endothelial cells. CD40 transmits signals for each of these cell types that regulates activation and differentiation (Hollenbaugh D. et al, EMBO J. 11: 4313–4321, 1992; Kiener P. A. et al, J. Immunol. 155: 4917–4925, 1995; Cella M. et al, J. Exp. Med. 184: 747–752, 1996; Galy A. H., and Spits H., J. Immunol. 152: 775–782, 1992; Clark E. A., and Ledbetter J. A., Proc. Natl. Acad. Sci. USA 83: 4494–4498, 1986). CD40 is activated by crosslinking during cell to cell contact with cells expressing CD40 ligand (CD154), primarily T cells. While soluble forms of CD154 can stimulate CD40, no attempts have been made to use or modify soluble CD154 to promote immune responses to antigens.
CD40 signals to B cells are required for isotype switching and affinity maturation through somatic mutation (Rousset F. et al, J. Exp. Med. 173: 705–710, 1991). In the absence of CD40 signals, germinal centers, the specialized sites of B cell maturation, are not formed, and B cells are unable to differentiate into IgG producing plasma cells (Foy T. M. et al, J. Exp. Med. 180: 157–163, 1994). Patients with HIGM syndrome are not able to form germinal centers or produce IgG antibodies after antigen challenge, and the same phenotype is seen in knockout mice where CD40 or CD154 is not expressed. The CD40 signal has been shown in vitro to promote survival of surface Ig-activated B cells, and to interact with signals from cytokines to induce immunoglobulin isotype switching to IgG, IgA, and IgE production (Holder M. J. et al, Eur. J. Immunol 23: 2368–2371, 1993; Jabara H. H. et al, J. Exp. Med. 177: 925–935, 1990; Grabstein K. H. et al, J. Immunol. 150: 3141–3147, 1993). In addition, HIGM syndrome patients and CD154 knockout mice have impaired lymphocyte proliferation in response to diphtheria toxoid, tetanus, and Candida, showing that the CD40 signal is required for T cell priming to protein antigens (Grewal I. S., and Flavell R. A., Annu. Rev. Immunol 16: 111–135, 1998; Toes R. E. M. et al, Sem. Immun. 10: 443–448, 1998; Grewal I. S. et al, Nature 378: 617–620, 1995; Ameratunga R. et al, J. Pediatr. 131: 147–150, 1997; Subauste C. S. et al, J. Immunol. 162: 6690–6700, 1999). Expression of CD154 in vivo to enhance immune responses utilized only the cell surface form of the molecule and resulted in significant toxicity in experimental animals, including induction of lethal autoimmune disease and T cell malignancies (Roskrow M. A et al, Leukemia Research 23: 549–557, 1999; Brown M. P. et al, Nature Medicine 4: 1253–1260, 1998).
In neonates, insufficient stimulation of CD40 due to low levels of expression of CD154 by activated T cells has been identified as a factor in the inability of infants to produce IgG antibodies towards bacterial antigens (Nonoyama S. et al, J. Clin. Invest. 95: 66–75, 1995; Fuleihan R. et al, Eur. J. Immunol. 24: 1925–1928, 1994; Brugnoni D. et al, Eur. J. Immunol. 24: 1919–1924, 1994). This suggests that CD40 signals are not ubiquitous and that highly restricted expression of CD154 may limit the extent of CD40 signaling and thus the magnitude and quality of an immune response. Direct evidence in support of this idea comes from a recent study where a modest increase (1.1–2 fold) in expression of cell surface CD154 in the thymus of mice resulted in a >10 fold increase in the antigen-specific antibody response (Prez-Melgosa M. et al, J. Immunol. 163: 1123–1127, 1999). Some evidence suggests that CD40 stimulation may be deficient in HIV-1 infected individuals, since HIV gp120 suppressed the expression of CD154 by activated T cells in vitro, and production of IL12 is defective in HIV-1 positive individuals (Chirmule N. et al, J. Immunol. 155: 917–924, 1995; Taoufik Y. et al, Blood 89: 2842–2848, 1997; Yoo J. et al, J. Immunol. 157: 1313–1320, 1996; Ito M. et al, AIDS Res. Hum. Retroviruses 14: 845–849, 1998; Benyoucef S. et al, J. Med. Virol. 55: 209–214, 1998). In addition, CD40 stimulation of dendritic cells infected with HIV-1 was found to suppress virus replication, suggesting that transmission of HIV-1 from infected dendritic cells during antigen presentation could be blocked by CD40 signals (McDyer J. F. et al, J. Immunol. 162: 3711–3717, 1999). However, a method for stimulation of CD40 on cells actively presenting antigen to T cells while avoiding toxicity from unregulated CD40 stimulation is needed.
CD40 signals to dendritic cells or B cells causes their differentiation from an antigen uptake function to an antigen processing and presentation function (Sallusto D. et al, J. Exp. Med. 182: 389–400, 1995; Cella M. et al, J. Exp. Med. 184: 747–752, 1996; Faassen A. E. et al, Eur. J. Immunol. 25: 3249–3255, 1995). This shift is accompanied by reduction of the MHC class II intracellular compartment, increased expression of MHC class II on the cell surface, secretion of the Th1 regulatory cytokine IL12 and increased expression of CD86 and CD80. After CD40 activation, dendritic cells and B cells are able to more efficiently present antigen and give a critical costimulatory signal through CD28. The production of IL12 leads to enhanced secretion of IFNγ by T cells and suppression of Th2 cytokine production. The CD40 signal is therefore an important mediator of Th1 cellular immunity and CTL induction. However, selective stimulation of CD40 during antigen presentation is needed to enhance immune responses to vaccination.
In addition to B cells and dendritic cells, CD40 is functionally active on other APC's such as monocytes, where CD40 signals prevent cell death from apoptosis and induce expression of adhesion molecules and production of inflammatory cytokines TNFα and IL8 (Kiener P. A. et al, J. Immunol. 155: 4917–4925, 1995). CD40 has also been reported to be expressed and functionally active on thymic epithelial cells (Galy A. H., and Spits H., J. Immunol. 152: 775–782, 1992) and on many kinds of tumor cells, including carcinomas, melanomas, and lymphomas (Ledbetter J. A. et al, In Leucocyte Typing III: White Cell Differentiation Antigens p. 432–435, 1987; Oxford University Press, Oxford, U.K.; Paulie S. et al, Cancer Immunol. Immunother. 20: 23–28, 1985). In contrast to most normal cells where the CD40 signal enhances survival, in many malignant cells CD40 actively promotes death by apoptosis. Therefore CD40 is functionally active in all cell types that express the receptor, and CD40 signals are central to fundamental processes of survival and differentiation. Because of the widespread expression of functional CD40, localized stimulation of CD40 positive cells that present specific antigen to T cells is desirable so that only APC involved in the specific immune response are activated.
Studies in CD154 knockout mice have confirmed the importance of CD40 activation for the antigen specific priming of T cells. CD154 deficient mice have an enhanced susceptibility to Leishmania major and Toxoplasma gondii infection, consistent with a central role for CD40 in cellular immunity (Subauste C. S. et al, J. Immunol. 162: 6690–6700, 1999; Campbell K. A. et al, Immunity 4: 283–289, 1996). CTL generation after viral infection in CD154 deficient mice is markedly blunted, and induction of experimental allergic encephalomyelitis (EAE) in response to myelin basic protein does not occur (Grewal I. S. et al, Science 273: 1864–1867, 1996; Grewal I. S. et al, 378: 617–620, 1995). The defect in T cell priming in these models appears to be due to an inability of APC to provide costimulatory signals to T cells (Grewal I. S. et al, Science 273: 1864–1867, 1996; Yang Y. and Wilson J. M., Science 273: 1862–1867, 1996).
Inhibition of CD40 in vivo has been studied in mice using a mAb, MR1, that binds and blocks the CD40 ligand, CD154 (Durie F. H. et al, Science 261: 1328–1330, 1993; Foy T. M. et al, J. Exp. Med. 178: 1567–1575, 1993; Foy T. M. et al, J. Exp. Med. 180: 157–163, 1994; Durie F. H. et al, J. Clin. Invest. 94: 1333–1338, 1994; Gerritsse K. et al, Proc. Nat. Acad. Sci. USA 93: 2499–2504, 1996). These experiments demonstrated that anti-CD154 prevents the induction of autoimmune diseases, including EAE after immunization with myelin basic protein, oophritis after immunization with zona pelucida antigen (ZP3), and spontaneous disease in lupus prone mice (Griggs N. D. et al, J. Exp. Med. 183: 801–807, 1996; Daikh D. I. et al, J. Immunol. 159: 3104–3108, 1997). Anti-CD154 was also effective in preventing both chronic and acute graft versus host (GVH) disease and in preventing rejection of heart allografts after transplantation (Larsen C. P. et al, Nature 381: 434–438, 1996). Thus, CD40 signals are required for T cell responses to antigen, and restriction of the CD40 signal with specific inhibitors is an effective method of limiting T cell priming during an immune response.
The CD40 receptor is therefore a proven target for regulation of antigen specific immunity. While biological inhibitors of CD40 have been studied extensively in mice and in nonhuman primates, there is a need for localized stimulation of CD40 on cells that present antigens to T cells in order to improve the effectiveness of vaccines.
Gp160, the product of the HIV-1 env gene, is cleaved in the Golgi complex into gp120 and gp41 proteins that remain associated through noncovalent interactions. Most neutralizing epitopes of the virus are located on gp120 and gp41, and are expressed by the intact env complex that has been shown to be a trimer (Kwong P. D. et al, Nature 393: 648–659, 1998). Monomeric gp120 can be released from the complex and expose immunodominant epitopes that are non-neutralizing and are located on the internal face of gp120 in the intact trimeric complex (Wyatt R. et al, Nature 393: 705–711, 1998; Broder C. C. et al, PNAS USA 91: 11699–11703, 1994). Thus, stabilization of the env complex is needed for an HIV-1 vaccine in order to preserve conformational epitopes important for neutralization and to mask immunodominant epitopes that are not relevant for neutralization of the env complex.
One attempt to produce a stable, properly folded gp120-gp41 complex was made by altering the cleavage site in gp160 between the gp120 and gp41 domains (Earl P. L. et al, J. Virol. 68: 3015–3026, 1994). By introducing a stop codon before the transmembrane domain of gp41, a soluble molecule composed of gp120 and the extracellular domain of gp41 was produced as a complex that folds properly to bind the CD4 receptor and to express some conformational epitopes. However, this molecule formed dimers and multimers rather than the stable trimers that comprise the native structure of the envelope glycoprotein as revealed in the crystal structure of the gp120 complex.
Three major sites of gp120 have been identified that are involved in cross-neutralization of diverse viral strains (Wyatt R. et al, Nature 393: 705–711, 1998). The V3 domain was found to express linear and conformational epitopes that can be recognized by antibodies that neutralize HIV-1. Although the V3 domain is a variable region, it contains a central portion shared by many HIV-1 isolates, particularly those found in the United States and Europe. The central portion has been called the principle neutralization epitope and is formed from a linear epitope of the amino acid sequence GPGRAF (SEQ ID NO: 28)(Broliden P. A. et al, Proc. Natl. Acad. Sci. USA 89: 461–465, 1992; Broliden P. A. et al, Immunol. 73: 371–376, 1991; Javaherian K. et al, Science 250: 1590–1593, 1990; Javaherian K. et al, Proc. Natl. Acad. Sci. USA 86: 6768–6772, 1989). Conformational epitopes of the V3 loop have also been identified that can be recognized by antibodies that are more broadly neutralizing.
The CD4 binding domain of gp120 is another neutralization site for antibodies directed to HIV-1 env. This domain is a nonlinear, conformational site that depends upon proper folding of gp120 (Kang C.-Y. et al, Proc. Natl. Acad. Sci. USA: 6171–6175, 1991; Lasky L. A. et al, Cell 50: 975–985, 1987). Antibodies can recognize distinct portions of the CD4 binding domain, and may have either type-specific or cross-neutralization properties (Pinter A. et al, AIDS Res. Hum. Retro. 9: 985–996, 1993). Although monomeric gp120 can retain CD4 binding function, a stable trimeric structure of gp120 is thought to be important for masking immunodominant epitopes that are expressed on the internal face of the intact complex (Wyatt R. et al, Nature 393: 705–711, 1998). A third domain of gp120 involved in virus neutralization is exposed upon binding to CD4, and functions to bind the chemokine coreceptor to allow virus entry into the cell (Rizzuto C. D. et al, Science 280: 1949–1953, 1998). Thus a stable trimer of HIV-1 env is needed to present the major cross-neutralization epitopes and to prevent exposure of internal, immunodominant epitopes that do not induce neutralizing antibodies.
CD154 is a TNF-related, type II membrane protein that forms stable trimers (Mazzei G. J. et al, J. Biol. Chem. 270: 7025–7028, 1995). Soluble fusion proteins of human CD154 have been expressed using murine CD8 at the amino terminal side of the CD154 molecule (Hollenbaugh D. et al, EMBO J. 11: 4313–4321, 1992). Single chain Fv (scFv) molecules have also been constructed using heavy and light chain variable regions cloned from the G28-5 hybridoma that produces antibody specific for human CD40 (Ledbetter J. A. et al, Crit. Rev. Immunol. 17: 427–435, 1997). Both CD154 and G28-5 scFv fusion proteins retain functional activity as soluble molecules in vitro. However, no use of these molecules to improve the effectiveness of vaccines has been found.